Bacteria colonization in tumor microenvironment creates a favorable niche for immunogenic chemotherapy

The tumor microenvironment (TME) presents differential selective pressure (DSP) that favors the growth of cancer cells, and monovalent therapy is often inadequate in reversing the cancer cell dominance in the TME. In this work, we introduce bacteria as a foreign species to the TME and explore combinatorial treatment strategies to alter DSP for tumor eradication. We show that cancer-selective chemotherapeutic agents and fasting can provide a strong selection pressure against tumor growth in the presence of bacteria. Moreover, we show that an immunogenic drug (oxaliplatin), but not a non-immunogenic one (5-FU), synergizes with the bacteria to activate both the innate and adaptive immunity in the TME, resulting in complete tumor remission and a sustained anti-tumor immunological memory in mice. The combination of oxaliplatin and bacteria greatly enhances the co-stimulatory and antigen-presenting molecules on antigen-presenting cells, which in turn bridge the cytotoxic T cells for cancer-cell killing. Our findings indicate that rational combination of bacterial therapy and immunogenic chemotherapy can promote anticancer immunity against the immunosuppressive TME.

13th Jul 2023 1st Editorial Decision 13th Jul 2023 Dear Dr. Mou, Thank you for the submission of your manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you.We have now received feedback from two of the three reviewers who agreed to evaluate your manuscript.As the referee #2 will unfortunately not be able to return his/her report in a timely manner, and given that both reviewers provide very similar recommendations, we prefer to make a decision now in order to avoid further delay in the process.
As you will see from their reports pasted below, both referees recognize potential interest of the study but also raise serious and partially overlapping concerns that should be addressed in a major revision.Particularly, in vitro co-culture experiments need to be significantly improved to support the conclusions on the mechanism.Should referee #2 provide a report, we will send it to you, with the understanding that we will not ask for an additional revision.If you would like to discuss further the points raised by the referees, I am available to do so via email or video.Let me know if you are interested in this option.
Further consideration of a revision that addresses reviewers' concerns in full will entail a second round of review.EMBO Molecular Medicine encourages a single round of revision only and therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
We would welcome the submission of a revised version within three months for further consideration.Please let us know if you require longer to complete the revision.
I look forward to receiving your revised manuscript.

Yours sincerely, Zeljko Durdevic
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Response:
We extend our gratitude to the reviewer for acknowledging the manuscript's quality and its relevance to the field of cancer therapy.Having meticulously reviewed our work, we acknowledged the disconnect between the concept of DSR and the later part that focused on tumor immune environment.As such, modification to the text and several display items have been made to harmonize the article.Details are as follows:  In the framework of DSR, we have revised the abstract and introduction to connect the immune status within the TME as an integral aspect of the DSR theory.In addition, we updated Figure 7 to highlight differential selective pressure in the TME in response to different combinatorial treatment strategies.
Several transition paragraphs are also provided in the body and discussion section to relate immune cell modulation to DSR principle.These transition paragraphs are as described below. "We hypothesized that achieving long-term tumor remission often relies on the engagement of immune responses, in particular the host adaptive immunity.However, within the tumor microenvironment (TME), tumor-infiltrating leukocytes tend to adopt a pro-tumoral phenotype.While introducing E. coli into the TME can activate a fraction of the immune system, the resulting selective pressure is non-specific and primarily targeting the bacteria rather than the tumor cells.To align with our concept of establishing DSR within the TME to target tumor cells, we then decided to explore an immunogenic drug with the ability to direct the host immune responses towards tumor cells."  Discussion section: "Solid tumors can be seen as ecosystems where the microenvironment is optimized for tumor cell growth.In ecological terms, tumor cells are the dominant species with limited competition from other members within the TME.In attempting to break this dominance, we introduced an invasive species (E.coli) into the TME to compete for survival resources.However, our experiment showed that bacteria monotreatment, although temporarily controlled tumor growth, could not achieve tumor eradication.Combinatorial treatments, such as 5-FU and fasting, were designed to selectively favor bacteria over cancer cells.While these combinatorial treatments enhanced tumor suppression, complete tumor remission remained elusive.Consequently, we modified the survival and selective factors in the TME by introducing an immunogenic drug, oxaliplatin, which in combination with bacterial therapy converts the immunosuppressive TME to an immunoreactive one [43,44]." 2nd Nov 2023 1st Authors' Response to Reviewers 2. The "fasting" experiment needs to be better embedded into the manuscript.It appears somehow disconnected!

Response:
We thank the reviewer for this suggestion.We have substantially revised the abstract, introduction, and Figure 7 to provide better justification for the "fasting" experiment.In short, we show that nutrient deprivation induces DSR that favors bacteria growth over cancer, and the combination treatment results in improved tumor suppression.We highlight that the anticancer enhancement due to fasting in vivo was modest due to practical limitations in executing nutrient deprivation in animals, and to facilitate transition of the study into the latter half of the section, we included additional paragraphs to justify design rationale towards combining bacterial therapy with immunogenic chemotherapy.The added paragraphs on page 7 are as follows: "Although the DSR provided by 5-FU, fasting, and E. coli significantly enhanced tumor suppression, the treatments were not able to achieve complete tumor remission.We hypothesized that achieving long-term tumor remission often relies on the engagement of immune responses, in particular the host adaptive immunity." 3. Concerning the translational aspects of the in vivo experiments, side-effects of the E. coli therapy need to be more closely monitored.What is the impact, especially of the intravenous bacteria application, on the immune conditions in the peripheral blood?What happens with systemic cytokine levels etc.? Is there any septic reaction and if not, please explain why?
Response: We thank the reviewer for the comment as safety stands as a paramount concern in any form of bacterial cancer therapy.
 Aligning with this principle, we selected a non-pathogenic E. coli strain (DH5α) as the bacteria treatment.The safety profiles of DH5α have been subject to comprehensive investigation [1].In addition, the safety of bacteria cancer therapy of non-pathogenic bacteria (such as E. coli DH5α/Nissile 1917) had been thoroughly assessed in mice model including survivability in blood, organs [2], serum cytokine profiles [3,4] and organ histology [5].All the safety assessments showed that the non-pathogenic strains of E. coli are safe to be administered intravenously. Furthermore, we assessed the safety profile of the bacteria by closely monitoring weight fluctuations and blood biochemical parameters in mice who received intravenous DH5α treatment.Mice received four-IV doses (1 x 10 8 E. coli/dose) showed no significant decrease in their body weight, indicating the dose applied is tolerable (Figure R1A; included in Figure S5B in the manuscript's supplementary information). For whole blood cells analysis.We observed a steep increase of neutrophils (43.56 %) at day 1 post IV inoculations of E. coli in comparison to the untreated control (26.2 %), indicating the first line immune defense against bacteria was activated.The effects persist till day 3 post inoculation and the readings fall back into the normal range at day-7 post inoculation (23.04 %) (Figure R1C; included as Figure S5E in the manuscript's supplementary information). In addition, we also conducted a hemolysis assay using mice RBC and assessed the effects of hemolysis by reading the A570 value of samples' supernatant for quantification of the release of hemoglobin into the solution after 30-min of co-incubation with E. coli (Figure R1B; included as Figure S5D in the manuscript's supplementary information).bacteria in 100 uL of PBS/dose) were given every 3 days throughout the treatment period.(B) Assessment of hemolytic potential of E. coli using murine RBC.Murine blood was extracted via cardiac blood collection.The blood was kept in EDTA tube followed by RBC isolation (900 xg centrifugation for 5 min and wash thrice with sterile PBS).The hemolysis assay was performed by incubating 4 X 10 7 E. coli with 1 mL of 1 % murine RBC at 37 0 C for 30 min.The resulting samples were then centrifuged at 900 xg for 5 min and hemolysis (release of hemoglobin) was determined by reading the absorbance of the at 570 nm.A negative control (treated with PBS) and a positive control (1% triton-X 100) were included.(C) Whole blood cell analysis of mice received 1-dose of IV-administrated E. coli (4 x 10 8 bacteria/dose).Blood samples were collected via cheek pouch method at Day-1, Day-3 and Day-7 post-treatment.
 In addition, in our co-culture study, we observed that DH5α can only proliferate normally in RPMI medium when heat-inactivated FBS was used, possibly due to the presence of complement system that could bind to the bacteria and lead to lysis of the bacteria.This observation was also confirmed by other independent studies: "In contrast, strains EQ1, DH5a, BLR and BL21were only able to survive in sera which had been heat inactivated and the numbers of viable bacteria were reduced to levels below detection by 30 min."[1].
4. The immune cell analyses in Figures 3 and 4 have obviously been done from the tumors at the end of the in vivo experiment, as exemplarily shown in Figure 2i.Does it make any sense to compare the immune compartments at this very late stage of therapy with minimal tumor volume especially in the combined treatment group.An experiment starting therapy with a more progressed tumor volume and then analyzing the tumor immune invasion during the response phase should be included.
 Response：We express our gratitude to the reviewer for raising this question.Our observations indicate that, at earlier time points (D1 and D3 post E. coli injection; tumor volumes were approximately 300-500 mm 3 at point of analyses), neutrophils predominate among the tumorinfiltrating immune cells, as illustrated in the figure below.It is worth noting that innate immunity typically responds rapidly, whereas adaptive immunity typically requires more time to reach its peak, with a lag of approximately 5-7 days post-exposure [6,7]. Given this understanding, we deliberately selected a later time point to assess the activation and infiltration status of T lymphocytes within the tumor.However, we also acknowledge that the infiltration patterns of immune cells at earlier time points provide valuable additional insights for our readers.Consequently, we are pleased to incorporate this data into the extended view (EV) section of the manuscript for the benefit of our audience (Figure R2; included as Figure S7C in the manuscript's supplementary information).5.The in vitro co-culture experiment is elegant but the inclusion of pen/strep to avoid E. coli overgrowth is kind of problematic.It has to be stressed that you see in these experiments primarily the effect of dead bacteria, which should be different in the in vivo setting.

Response:
 We appreciate the reviewer's positive feedback, and in response, we would like to present some preliminary data to provide a rationale for our choice of using PenStrep (dead bacteria) instead of live bacteria in the in vitro experiments.We performed some additional studies to compare the effects of living and dead bacteria towards spleenocytes in an identical co-culture system. Due to the strong proliferative ability of E. coli, we inoculated 10 X fewer E. coli (2 X 10 5 cells for live E. coli) and shortened the incubation period from 48 h to 24 h (MOE_L).However, even under these modified conditions, we still observed overgrowth of live E. coli, characterized by slightly turbid medium and a color shift from red to orange.Subsequently, we performed flow cytometry analysis to assess the composition of spleenocytes that were co-incubated with MC38, E. coli, and oxaliplatin. Interestingly, we noted a higher proportion of dead spleenocytes compared to their PenStrep-treated counterparts.This difference can be attributed to the intense nutrient competition among the three components in the culture medium.However, despite these viability disparities, our analysis of immune cell activation patterns revealed consistent findings.Both the groups with live or dead E. coli exhibited (as Figure R3; included as Figure S12 in the manuscript's supplementary information): 1. Upregulations of the CD25 marker in CD4+ and CD8+ T-cells.
3. Enhanced MHCII expression on all antigen-presenting cells. Based on these observations, we rationalize that the addition of PenStrep would not alter the main conclusions of our in vitro spleenocyte assays.Additionally, it helps to mitigate the complications introduced by bacterial overgrowth and nutrient competition, ensuring the reliability and consistency of our in vitro assay outcomes.6. Legend of the color codes in Figure 4e are missing!Response: We thank the reviewer for bringing this matter to our attention.We have now reintroduced the color codes to the figure as per your suggestion.
7. Statistics in Figure 5a are missing and generally a chapter on statistics would be desirable.
Response: We extend our gratitude to the reviewer for addressing this concern.In response, we have reexecuted the experiment and reconstructed Figure 5 to display the absolute counts of each spleenocyte population.This modification serves to provide a more accurate representation of the proliferation of each spleenocyte population in our study, in line with the professional suggestion made by the other reviewer.Additionally, we have incorporated a paragraph in the methodology section that delineates the statistical analysis parameters employed in this study for clarity and transparency.
"All statistical analyses were done using GraphPad Prism 8.0.Statistical differences among different treatment groups of all tumor-growth curve were computed using 2-way ANOVA analysis (when groups > 3) with Tukey's multiple comparisons test while paired t-test (two-tailed) was used to compute statistical difference between two growth curves (group = 2).On the other hand, statistical differences between different column groups were computed using 1-way ANOVA (when groups > 3) with Tukey's multiple comparisons test whereas unpaired t-test (2-tailed) were applied for comparison between two column groups." 8. How can tumor-specific viability be seen in the CCK8 assay when viable splenocytes (and probably even some living bacteria) are present?Please explain!

Response:
We appreciate the reviewer's query, and we would like to offer clarification on this matter by presenting some of our preliminary data obtained during assay optimization.
 Before conducting the CCK8 assay, we performed two washes with PBS in each well.This step was taken because most bacteria and spleenocytes are non-adherent, except for those adhered to the surface of MC38. Furthermore, it is important to note that signal changes in CCK8 assays primarily depend on the metabolic activity of the cells (conversion of WST-8 dye into colored formazan by cellular dehydrogenases) under investigation.In preparation for the assays, we carried out an analysis using spleenocytes alone to assess their reactivity to the CCK8 reagent.This analysis revealed that the metabolic activity of spleenocytes was considerably slower compared to that of MC38 cells.In Figure R4A (included as Figure S13C in the manuscript's supplementary information), we have included a standard curve depicting the relationship between the number of spleenocytes and the A450 signal derived from the CCK8 assay.
 Additionally, we presented the normalized absorbance values from the co-culture assays, assuming an A450 value equivalent to 5x10 4 spleenocytes (assuming no spleenocytes were washed away).It is evident that the contribution of spleenocytes to the A450 signal was minimal, and as such, it should not significantly impact the results of the CCK8 assay.(as Figure R4; included as Figure S13B and S13C in the manuscript's supplementary information):

Response:
We are grateful to the reviewer for pointing out this matter, and we sincerely apologize for the omission in the manuscript body.The supplementary figures resulting from this concern have been appropriately described and integrated into the manuscript as follows:  "However, an additional assay comparing the number of tumor-harboring E. coli showed no significant differences between mice that received only E.coli or E.coli and oxaliplatin, indicating the in vivo dosage applied in our study did not affect the survival and colonizing ability of E. coli (Fig S11a and b)."

Referee #3 (Remarks for Author):
In their manuscript, Lim et al. present data on bacteria mediated cancer therapy.They use murine transplantable colon carcinoma (mainly M38 in C57Bl/6 mice), a laboratory strain of E. coli and co-treat the tumor bearing mice with 5-FU or oxaliplatin.The combined treatment of the cancer bearing mice with E. coli and oxaliplatin shows in some cases complete clearance of the tumors.The authors present then data on tumor infiltration by cells of the innate immune system and their differentiation status as well as the infiltration by cells of the adaptive immune system.This part of the manuscript is highly interesting and the reason for my positive evaluation of the manuscript although some weak point need to be ruled out first.The authors then try to show the mechanism of the effect of co-administration by in vitro coculture.This part is too preliminary and not convincing at all.It is completely over interpreted and technically extremely weak.It should be deleted from the manuscript and worked out in more details and more convincingly.In details: 1. Results: it should be pointed out that the administration of the bacteria was done intratumorally.How often the bacteria were applied should also be mentioned when appropriate.

Response:
We thanked the reviewer for raising this issue, we had added in the administration route of E. coli and the chemotherapeutics in the Figure legend 1(c) and 2(c).

 "1(c) In vivo anti-tumor activity of PBS, 5-FU, E. coli, and 5-FU+E. coli in the MC38 syngeneic murine model, 5-FU and E. coli were delivered intraperitoneally and intratumorally respectively.""2(c) In vivo anti-tumor activity of PBS, OXA, E. coli and OXA+E. coli in the MC38
syngeneic murine mode, OXA and E. coli were delivered intraperitoneally and intratumorally respectively." We also stated the dosing of bacteria in the methodology section under the title "In vivo Anti-tumor Assays of 5-FU" and "In vivo Anti-tumor Assays of Oxaliplatin".
2. Results: the Kaplan Meier blot in Fig. 1h shows no difference between 5-FU +E and 5-FU+E+starvation.In the text a difference is claimed.

Response:
We thanked the reviewer for raising this issue and we apologize for the confusion of the graph.We had remade the Kaplan Meier plot to provide better clarity of the study.The figure is included as Figure 1H in the revised manuscript. At the conclusion of this study, two-third of the mice in the starvation + 5FU + E treatment group survived the assay using tumor volume = 1500 mm 3 as a cutoff point.In contrast, all mice in the solvent group and 5FU+E were determined to reach the endpoint of the study at day 14 and 16 respectively.
3. Results: why was starvation not tested for Oxa+E.coli?
Response: We express our gratitude to the reviewer for bringing up this question, and we recognize our previous manuscript did not properly justify the inclusion of starvation and its subsequent exclusion in the Oxa+E.coli treatment.To place the starvation study in its proper context, we have substantially modified the abstract, introduction, background, discussion, and Figure 7 to harmonize the different segments of the study.In short, inclusion of nutrient deprivation was designed to highlight strategies to enhance survival advantage of bacteria over cancer in different treatment combinations.In short, we show that nutrient deprivation induces DSR that favors bacteria growth over cancer, and the combination treatment results in improved tumor suppression.We point out that the anticancer enhancement due to fasting in vivo was modest due to practical limitations in executing nutrient deprivation in animals, and to facilitate transition of the study into the latter half of the section, we included additional paragraphs to justify design rationale towards combining bacterial therapy with immunogenic chemotherapy.The added paragraphs on page 7 are as follows: "Although the DSR provided by 5-FU, fasting, and E. coli significantly enhanced tumor suppression, the treatments were not able to achieve complete tumor remission.We hypothesized that achieving long-term tumor remission often relies on the engagement of immune responses, in particular the host adaptive immunity." Furthermore, it's worth noting that the combination of oxaliplatin and intratumorally delivered E. coli has demonstrated the capability to completely eliminate tumors in our mouse model.Consequently, we decided to discontinue the starvation assay for the oxaliplatin-based treatment studies for both narrative and ethical reasons.We believe the revised manuscript better justifies our study design and appreciate the reviewer's suggestion.
4. Results: Fig. 2l.The same spleen size is claimed for O and OE but 59mg compared to 89mg is for me not the same.Response: We express our gratitude to the reviewer for highlighting this issue and allowing us to clarify it.Our primary intent is to highlight that the combination of oxaplatin and E. coli reduced spleen enlargement, and we did not mean to claim that O and OE groups had the same spleen size.As such, we have revised the description text in the manuscript body to better describe this observation: "Surprisingly, the OXA+E.coli co-treatment rescued the spleen size to a normal range in all tested mice (N=5), suggesting a reduction bacteria-associated systemic reactogenicity by the OXA combination." 5. Results: in general, all flow cytometry data are displayed as percentage.In some cases, this might still be meaningful but only in combination with absolute numbers all these data are acceptable. Response: We appreciate the valuable suggestions from the reviewer.Upon careful consideration of our data, we concur that utilizing absolute counts to analyze the flow cytometry data for in vitro assessment of spleenocyte composition provides a more direct and informative perspective regarding the stimulation (proliferation) or elimination of spleenocytes by each treatment.Consequently, we have taken the decision to repeat the experiment and present the results in the form of absolute counts, which we will address in subsequent points. Regarding the in vivo analysis of Tumor-Infiltrating Lymphocytes (TILs), we understand and share your concerns and therefore, we included the flow cytometry data in the form of cell count in our supplementary data (S11).We maintained the data presentation in percentages in the manuscript body as the initial tumor volumes processed for TILs analysis can exhibit significant variation, especially when comparing combinations (OE) and the control (PBS) treatments, where the difference in tumor volume could exceed tenfold (e.g., 42 mg vs. 1820 mg).In our view, presenting the flow cytometry data in the form of percentages for the in vivo assays serves as a normalization method that helps account for differences in the initial sample sizes being processed.This approach helps mitigate the impact of varying tumor volumes on the analysis and allows for a more accurate assessment of treatment effects on TILs.
6. Results: although OXA depletes all tumor infiltrating immune cells, the authors blame on the cytotoxicity of Oxa.Why do those cells survive when E. coli are co-administered?This needs to be explained either here or in the discussion.
 We appreciate the reviewer's question, and we have included in the discussion our hypotheses to explain the observed scenarios:  Immunostimulant Compensation: The addition of E. coli, a potent immunostimulant, may stimulate the proliferation of immune cells, thus compensating for the loss of immune cells due to the cytotoxic effects of chemotherapy.This hypothesis is supported by data from our in vitro spleenocyte analysis.Among all the cell types analyzed, antigen-presenting cells (APCs), including B cells, dendritic cells (DC), and macrophages, were relatively more sensitive to the cytotoxic effects of oxaliplatin.For example, the count of B cells decreased from 44,050/mL (in the M group) to 29,418/mL after 24 hours of incubation with 2 uM of oxaliplatin.However, the addition of E. coli partially mitigated these effects, resulting in a B cell count of 39,424/mL in the MOE group (Figure R3; included as Figure 5A in the manuscript's body).Similar trends were observed in the DC and macrophage subsets.This hypothesis suggests that E. coli acts as a supportive factor in maintaining and stimulating certain immune cell populations, counteracting the cytotoxicity induced by oxaliplatin.Further analysis may be necessary to explore the mechanisms underlying this compensatory effect and its relevance to the observed outcomes in our study. Immune cells recruitment: Furthermore, in an in vivo system, the presence of Tumor-Infiltrating Lymphocytes (TILs) is not solely dependent on the proliferation or maturation of existing immune cells in the tumor but is also influenced by the infiltration of immune cells from distant sites, such as those from draining lymph nodes or the spleen.The Pathogen-Associated Molecular Patterns (PAMPs) from E. coli and Damage-Associated Molecular Patterns (DAMP) from oxaliplatin-treated MC38 both served as are potent immune attractants that are capable of stimulating the proliferation of immune cells in secondary lymphoid organs and facilitating their migration into the tumor microenvironment (TME). This additional mechanism underscores the multifaceted impact of E. coli in our study, as it not only supports the maintenance and proliferation of immune cells within the TME but also promotes the recruitment of immune cells from peripheral sources, contributing to the observed increase in TILs.
Further investigations could delve into the precise pathways and signaling mechanisms involved in this immune cell mobilization and infiltration, thereby enhancing our understanding of these complex interactions.7. Results: Fig. 4a-d.Only TNF-a and IFN-g is used as activation marker.Additional markers including other activation markers need to be included in the analysis.Response: We thank the reviewer for the valuable comment.While we acknowledge that a wide arrays of T cell activation markers (i.e.CD25, CD44, CD107a, CD137…etc) could add to the rigor of the tumor infiltrating lymphocyte analysis, we would like to clarify that for the purpose of the present article, TNF-a and IFN-g are sufficient in portraying the increasing number of functional tumor infiltrating lymphocytes in the TME following oxa+E.coli therapy.In conjunction with Figure 2F that proves anticancer immunological memory and Figure 4A, B that show heightened CD4 and CD8 populations in the tumor of oxa+E.coli-treated mice, the putative functional markers of TNF-a and IFN-g add to our overarching narrative of enhancing immune cell populations to exert differential selective pressure on cancer cells.We acknowledge that our original manuscript had a liberal use of "activation" when describing TILs, which led the reviewer to seek for additional activation markers.As such, we revised the descriptions to "functional" T cells when describing TNFa+ and IFNg+ CD8 T cells in the TME. Response: We thank the reviewer for raising this issue, the color codes were added back to the figure.9. Results in vitro: as pointed out already, these data are extremely preliminary.Fig. 5.These data are meaningless unless the absolute numbers are shown.Numbers of innate immune cells like DC or macrophages increase.Are the proliferating?This is unlikely since they are considered to be end cells.Under some conditions, they might proliferate.This needs to be shown.Why was isolated LPS not tested?
Response: The objective of the in vitro assay is to supplement our in vivo data by investigating the potential interactions among each treatment arms in a controlled environment and provide a hypothesis of how those interactions contribute to the cancer cell cytotoxicity.Our in vitro data showed that E. coli is responsible for the activation of immune cells (both APCs and T-cells; Figure R4B, C and D; included as Figure 5B, C and D in the manuscript body respectively) while oxaliplatin bridged the activated immune cells to cancer cell cytotoxicity (Figure R4E; included as Figure 5E in the manuscript body).We appreciate the reviewer's guidance and have taken the necessary steps to address the concerns raised:  Presentation of In Vitro Spleenocyte Assays: We have re-conducted the in vitro spleenocyte assays and have now presented the data in Figure R4 in the form of cell number/mL (included as Figure 5 in the manuscript body), as suggested. Dendritic Cells (DC) and Macrophages: While DCs and macrophages may not typically proliferate in this context, it is indeed possible for monocytes to undergo maturation and transformation into DC or macrophage-like cells, as indicated by the upregulation of maturation markers such as CD11c and F4/80 upon oxaliplatin treatment in the presence of LPS.We appreciate the reference provided to support this observation [8]. Cytotoxicity Assay: We conducted a cytotoxicity assay by co-incubating different concentrations of LPS with oxaliplatin and MC38.However, we observed no significant difference in oxaliplatininduced cytotoxicity, even at high LPS concentrations (1000 ng/mL; typical in vitro dosage = 1-100 ng/mL).(Figure R5A; included as Figure S13D in the manuscript's supplementary data). Notably, the addition of whole bacteria (MOI = 10) led to a twofold increase in oxaliplatin potency.
This finding suggests that the synergistic effect between E. coli and oxaliplatin is likely attributed to factors beyond LPS, such as other Pathogen-Associated Molecular Patterns (PAMPs) from the bacteria, including flagellin, CpG ODN, peptidoglycan, and more.These additional PAMPs may play a crucial role in enhancing the combined therapeutic efficacy of E. coli and oxaliplatin.(Figure R5B; included as Figure S13E in the manuscript's supplementary data).
 We believed that these adjustments and explanations will address the concerns raised and provide a clearer understanding of our research findings.10.Results: the authors then investigate the reactivity of spleen cells from naïve mice towards the tumor cells.First of all, it is not clear how they determine cytotoxicity or inhibition in the mixture of tumor and spleen cells.Then they claim that the spleen cells specifically attack the tumor cells when Oxa and E coli are in the co-culture.They try to show this by pictures o aggregates between tumor cells and spleen cells after two days of co-culture without staining the T cells.Do the authors really believe that that many tumor specific T cells are in the spleen of a naïve mouse?
Response: We thank the reviewer for raising this issue.We would like to clarify this issue by showing some of our preliminary data while performing assays optimization and provide some explanations to the questions raised.
 Whether the CCK8 signals originated from tumor or spleen cells: o Prior to CCK8 assay, each well was washed thrice with PBS as most bacteria and spleenocytes are non-adherent (except for those who adhered to the surface of MC38).o In addition, signal changes in CCK8 assays rely largely on the metabolic activity of the cells studied.Prior to the assays, we conducted an analysis using only spleenocytes to determine its reactivity to the CCK8 reagent.The metabolic activity of the spleenocytes were significantly slower compared to the MC38 and in Figure R6 we showed a standard curve of number of spleenocytes versus A450 signal from the CCK8 assay.We also showed the normalized absorbance values of the co-culture assays with A450 value of 5x10 4 spleenocytes (assuming no spleenocytes were washed away) where the A450 contributed by the spleenocytes were very low and it should not interfere with the results of the CCK8 assay (as Figure R6; included as Figure S13B and S13C in the manuscript's supplementary information).o We agree that the spleen of a naïve mouse should not primarily contain tumor-specific T cells.Our hypothesis is that the observed killing effects of the spleenocytes result from the collaborative efforts of both innate and adaptive immune cells.o We do not suggest that all the spleenocytes adhering to the MC38 cancer cells are CD8+ T cells.
Instead, it's likely a combination of activated phagocytes such as macrophages and dendritic cells, along with T cells, actively sampling antigens presented by these phagocytes.o When MC38 is treated with oxaliplatin, it can induce immunogenic cell death, leading to processes such as the surface exposure of calreticulin and the release of HMGB1 and ATP, which can attract antigen-presenting cells (APCs) to bind to the cancer cells [9,10].Furthermore, our flow cytometry data show that activated APCs upregulate their antigen-presenting capacities (MHC-II) and co-stimulatory receptors (CD80 and CD86).This upregulation could enhance APC-T cell interactions, increasing the likelihood of T cell antigen sampling from APCs.Therefore, it's 11.Discussion: it is not true that bacteria do not support the adaptive immune response against tumors.This has been shown in several publications.

MS
 Response: We appreciate the reviewer's feedback and have dialed back our tune in the revised manuscript.We have removed statements indicating that bacteria does not support the adaptive immune response against tumors.In the revised manuscript, we highlight that the combination of bacteria and immunogenic chemotherapy can bolster tumor-reactive adaptive immune responses.
12. Supplement: in general, all the flow cytometry panels are too small.The writing here is most of the time unreadable. Response: We thank the reviewer for pointing out this issue, we had remade the graphics in landscape format and provided images with better resolution to ensure the labels on the flow cytometry panels are readable upon enlargement of the figures.Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.We have now heard back from the two referees who agreed to re-evaluate your manuscript.As you will see from the report below, while the referee #1 is supporting publication of the manuscript, the referee #3 acknowledges the improvements of the revised manuscript but also raises some concerns that should be addressed in additional and final round of revision.Specifically, please consider removing the figures and results as indicated in point #3 and point #7.
Acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.
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Currently, our records indicate that the ORCID for your account is 0000-0002-0988-7029.In this revised version, Lim et al. describe the synergy of Oxaliplatin and E. coli for tumor therapy.The revision has significantly improved the manuscript.Nevertheless some points are still problematic.In detail (note minor and major points are mixed): 1. English: in general English is good but at some locations it needs to be corrected, like: 1 line Introduction:...comprisising of cancer cells..., or Introduction:...mice models...etc.2. Results: it needs to be mentioned at the beginning of the results that the application of E. coli is carried out intratumorally .This is important because in most publication bacteria are applied i.v. 3. Results: the starvation together with FU and E. coli does not any significant effect on tumor volume.Nevertheless the authors show a Kaplan-Mayer-Blot where the survival which is based on tumor size and by which the authors try to proof a high efficacy of the combinatorial treatment.However, they stop the experiment at day 16.Thus it is not clear which size the tumors would have had a day later.Since the experiment is apparently carried out only once, it is also not clear how reproducible the finding is.Anyway, I do not understand why the authors display this experiment since in the subsequent experiments they do not apply this condition.It should be deleted as figure and in the text.4. Results: the authors claim that the immune response in tumors experimentally colonized by bacteria is mainly directed against the bacteria.I am not aware of a study showing this. 5. Results: The authors claim that the absence of enhancement of T cell infiltration in Tumors treated with FU+E. coli is expected, but quote only papers on FU. 6. Results: in the rechallenge experiments, the authors do not mention after which time the rechallenge was done.No control unrelated tumor was applied.Although, are most likely corrected they cannot exclude the mice were in a general inflammatory state that does not allow to implant subcutaneously a tumor.7. Results: the increase of cytotoxicity when spleen cells are added is not very impressive and I am not convinced that it is meaningful.How often was the experiment done?What are the ideas of the authors about the mechanism that provides the additional cytotoxicity.

Response to Editor:
1) Figures: -We note that some images/panels are reused.Figure 2D is reused in Appendix Figure S3 and Figure 6D in Appendix Figure S14.Please cite in the respective figure legend every reused image/panel.
-In figures where n=2 please show raw values from both measurements and remove error bars.When n=2 statistical analysis is not recommended and a justification for the use of the statistical test employed has to be provided.Please check our Author Guidelines: https://www.embopress.org/page/journal/17574684/authorguide#statisticalanalysis

Response:
 We thank the editor for the information.We had included the respective description in the legends of Appendix Figure S3 to indicate that part of the figures was presented in the main text. "Part of the figures were presented in Figure 2D." For Figure 6D, the figure was removed from the manuscript according to the reviewer's advice. For figures with n = 2, we had removed all the error bars and present the data as individual plot for easy visualization.
2) In the main manuscript file, please do the following: -Please address all comments suggested by our data editors listed below: o Please note that the error bars are not defined in the legend of figures 1a-c, e-g; 2a-c, f-h; 3a-j; 4a-d, f-h; 5a-e; 6a-b.

Response:
 The following sentence is added into the figure legend for definition of the data plot and error bar: o For Figure 1  o Please note that the figure legend style does not comply with the journal guidelines i.e. all the figure legends are in a run-on style.
 We had reformatted all the figure legends in the main text to comply with the journal requirement.An example of the reformatted legend is as follow: 18th Dec 2023 2nd Authors' Response to Reviewers

"Figure 1. 5-FU or starvation provided differential selection pressure against cancer cells over bacteria.
A. In vitro cytotoxicity of 5-FU against the CRC cancer cell line (MC38; n = 3).B. In vitro cytotoxicity of n = 3).C. In vivo anti-tumor activity of PBS, E. coli, in the MC38 syngeneic murine model, 5-FU and E. coli were delivered intraperitoneally and intratumorally respectively (n = 5 mice for each treatment group).D. Kaplan-Meier analysis of mouse survivals in (C).
Data information: Mice were considered dead when the tumor volume exceeded 1,500 mm 3 .All curves are presented as mean with SEM as error bars and all column plots are presented as individual value with SEM error bars.Statistical differences among tumor-growth for more than 2 groups were computed using two-way ANOVA with Turkey's multiple comparison test.The differences were considered statistically significant when p-value < 0.05 in all statistical analysis.Significance symbols are defined as * p <0.05; ** p = 0.01 -0.05; *** p = 0.0001 -0.001; and **** p < 0.0001." o Please note that a separate 'Data Information' section is required in the legends of all the figures.
 A separate data information section is included in the legends of Figure 1   Statistical callout had been included in Figure 2H.However, Figure 1H have been removed from the manuscript body according to reviewer suggestion.For Figure 3F, callout was initially included in the figure, but it was taken out in accordance to the manuscript policy that suggests statistical analysis should not performed when n=2 (as in point no.1).
-In M&M, provide the antibody dilutions that were used for each antibody.
 We had included the dilution factors for all antibodies listed in the material and method section.An example is as follow:  "eFluor™ 780 eBioscience™ Fixable Viability Dye (Thermofisher Scientific;Cat# 65-0865;1: 2000)" -Please rename "Conflict of Interest" to "Disclosure Statement & Competing Interests".We updated our journal's competing interests policy in January 2022 and request authors to consider both actual and perceived competing interests.Please review the policy https://www.embopress.org/competing-interests and update your competing interests if necessary.
 We had renamed the section to Disclosure Statement & Competing Interests as requested.
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3) Appendix: Please add table of content with page numbers and correct nomenclature for Appendix Figure S1 etc., also in the main text.
 A table of content was added as requested and all the figures in appendix were renamed into Appendix Figure 1 … 16 accordingly, in the main text and appendix section.
4) Funding: Please make sure that information about all sources of funding are complete in both our submission system and in the manuscript.Currently there is a discrepancy between entry in our submission system and manuscript: National Science and Technology Council of the Republic of China in the manuscript file and Ministry of Science and Technology, Taiwan (MOST) in our system.Please correct.
 We apologize for the misunderstanding.There is a recent change in the naming of the funding agency to National Science and Technology Council of the Republic of China.
We had made the relevant correction in the manuscript submission system.
5) The Paper Explained: Please provide "The Paper Explained" and add it to the main manuscript text.Please check "Author Guidelines" for more information.https://www.embopress.org/page/journal/17574684/authorguide#researcharticleguide  A "The Paper Explained" section is added into the end of the main text, addressing the problem, result and impact of the study.-Synopsis text: Please provide a short standfirst (maximum of 300 characters, including space) as well as 2-5 one sentence bullet points that summarise the paper as a .docfile.Please write the bullet points to summarise the key NEW findings.They should be designed to be complementary to the abstract -i.e.not repeat the same text.We encourage inclusion of key acronyms and quantitative information (maximum of 30 words / bullet point).Please use the passive voice.-Please check your synopsis text and image before submission with your revised manuscript.Please be aware that in the proof stage minor corrections only are allowed (e.g., typos).
 A synopsis figure and word file with synopsis text is included along with the submitted manuscript.) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.Please note that the Authors checklist will be published at the end of the RPF. Response:  We agree to the publication of the RPF files.9) Please provide a point-by-point letter INCLUDING my comments as well as the reviewer's reports and your detailed responses (as Word file).
 We also added a descriptive text in the main text to highlight the administration route of the bacteria treatments. "All bacteria treatments were administered intratumorally unless otherwise specified." 3. Results: the starvation together with FU and E. coli does not any significant effect on tumor volume.Nevertheless the authors show a Kaplan-Mayer-Blot where the survival which is based on tumor size and by which the authors try to proof a high efficacy of the combinatorial treatment.However, they stop the experiment at day 16.Thus it is not clear which size the tumors would have had a day later.Since the experiment is apparently carried out only once, it is also not clear how reproducible the finding is.Anyway, I do not understand why the authors display this experiment since in the subsequent experiments they do not apply this condition.It should be deleted as figure and in the text.
 Response:  We appreciate the reviewer's comments.After a thorough review of our manuscript, we have decided to relocate the starvation results to the supplementary data.Additionally, we have revised the relevant paragraph to emphasize the limitations of the in vivo starvation approach, providing a more accurate reflection of the results.We also offer explanations to the audience regarding the decision not to continue with starvation in the subsequent parts of the study.
4. Results: the authors claim that the immune response in tumors experimentally colonized by bacteria is mainly directed against the bacteria.I am not aware of a study showing this.

Response:
 We thank the reviewer for raising this question, we acknowledged the ambiguity in the sentence, and we made the following amendment.o From: "the resulting selective pressure is unspecific and primarily targets the bacteria rather than the tumor cells" o To: "the resulting selective pressure is non-tumor specific"  The statement above refers to publications highlighting the efficacy of bacteria therapy, indicating that bacteria administration into tumors mainly activates strong infiltrations of innate immune cells such as neutrophils (Westphal et al., 2008) and macrophages (Weibel et al., 2008).These infiltrations lead to both the necrosis of the tumor tissue, bacteria clearance at the proliferating tumor rim as well as containment of the bacteria in the necrotic zone of the tumor.The strong infiltration of innate immune cells could result in temporally suppression of the tumor but also limit the long term therapeutic efficacy of the bacteria monotherapy. These observations align with our in vivo data, where synergism was observed only between the anti-tumor efficacy of E. coli and oxaliplatin.Tumor-infiltrating lymphocyte (TIL) analysis also revealed the highest proportion of neutrophils in the group receiving E. coli monotherapy, but not in the combined regimen where very limited numbers of neutrophils were determined and CD8+ T cells dominated the tumor microenvironment (TME).

Results:
The authors claim that the absence of enhancement of T cell infiltration in Tumors treated with FU+E. coli is expected, but quote only papers on FU.

Response:
 We thank the reviewer for raising this question, we acknowledged the ambiguity in the sentence, and we made the following amendment to ensure clarity of the statements being claimed.
From: "It is worth noting that 5-FU is a low-immunogenic agent as it could not efficiently induce ICDs in cancer cells (Vincent et al., 2010;Yamamura et al., 2015).Indeed, we found no enhancement of CD4 + or CD8 + tumor-infiltrating lymphocytes (TILs) in the group of 5-FU+E.coli co-treatment when compared to the other three treatment groups (Fig. S3)." To: "Examination of the TME of the 5-FU+E.coli group showed no enhancement of CD4 + or CD8 + tumor-infiltrating lymphocytes (TILs) (Appendix Figure S3), indicating that the treatment combination was ineffective in elevating anticancer adaptive immunity (Vincent et al., 2010;Yamamura et al., 2015)." 6. Results: in the rechallenge experiments, the authors do not mention after which time the rechallenge was done.No control unrelated tumor was applied.Although, are most likely corrected they cannot exclude the mice were in a general inflammatory state that does not allow to implant subcutaneously a tumor.

Response:
 We appreciate the reviewer for bringing up this concern and apologize for overlooking the details of the rechallenge in the manuscript.We have now included the relevant information on the rechallenge experiment in the methodology section as follows: o "The rechallenge was performed at least 3 weeks after the last administration of E. coli and two weeks after no tumor growth was observed in mice." While we acknowledge the possibility raised by the reviewer, we consider it unlikely for the general inflammation stage to persist for such a prolonged period that hinders tumor growth.Moreover, studies have indicated that chronic inflammation promotes all stages of tumorigenesis.
7. Results: the increase of cytotoxicity when spleen cells are added is not very impressive and I am not convinced that it is meaningful.How often was the experiment done?What are the ideas of the authors about the mechanism that provides the additional cytotoxicity.
 We appreciate the reviewer's feedback and after careful consideration, we decided to move the relevant figures into the supplementary data section. The experiment was done twice (biological replicate) with at least three replicates for each data point.We hypothesized that the additional toxicity from spleenocytes could be attributed to the following reasons: o Oxaliplatin induce immunogenic cell death in MC38, leading to processes such as the surface exposure of calreticulin and the release of HMGB1 and ATP, which can attract antigen-presenting cells (APCs) to bind to the cancer cells (Alcindor & Beauger, 2011;Stojanovska et al., 2019;Tesniere et al., 2010).o APCs such as macrophages could be activated/polarized into M1-state that could exert their tumoricidal activity via secretion of reactive nitrogen/oxygen species as well as proinflammatory cytokines (Boutilier & Elsawa, 2021;Guo et al., 2017;Li et al., 2021).o Direct cytotoxicity from APC-primed T-cells to MC38 cells.o We also conducted confocal microscopy experiments to identify the types of immune cells that were attached to the MC38 cells.Our observations revealed that dendritic cells, We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.
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Figure R2 .
Figure R2.Composition of the tumor infiltrating immune cells in each treatment group.For mice received E. coli treatment (IT), three end points were applied for tumor harvesting and immune cells profiling (Day 1, 3 and 8 after the last dosage of E.coli administration).

Figure R3 .
Figure R3.Comparison of the effects of in vitro spleenocytes activation using live (no PS; MOE_L) versus growth-inhibited bacteria (with PS; MOE).(A) Percentage of dead spleenocytes 24 hours posttreatment.(B) Percentage of activated T-cells (CD25+) 24 hours post-treatment.(C) Percentage of CD80+/86+ population for each type of APCs at 24 hours post-treatment.(D) Percentage of MHCII+ population for each type of APCs at 24 hours post-treatment.

Figure R4 .
Figure R4.Assesment of CCK-8 signals for murine spleenocytes.(A) Standard curve demostrating colleration between spleenocyte counts with the CCK-8 signals (A450).Spleenocytes were counted using hemocytometer under microscope prior to the generation of standard curve.(B) Raw absorbance data for the MC38-spleenocyte co-culture assay.In the co-culture assay, 5 x 10 4 spleenocytes were added to each well and the corresponding CCK-8 signals were ploted on the graph with assumption of none of the spleenocytes were washed away during the washing steps.

Figure R1 .
Figure R1.Kaplan Meier plot remade for Figure 1 (D) in the manuscript body.

Figure R2 .
Figure R2.Spleen weight of each treatment group at Day 15 post-treatment (Figure 2G; n = 5 for each treatment group and n = 4 for healthy control).

8 .
Results: Fig. 5e.Legend is missing.It is completely unclear what is shown here.

Figure R4 .Figure R5 .
Figure R4.The cancer/immune/bacteria co-culture system reveals the synergistic interaction of OXA and E. coli for immune cell activation.The immune cells (murine splenocytes) and the cancer cells (MC38) were co-cultured and subject to various treatments (NT: splenocytes alone.M: splenocytes + MC38.ME: splenocytes + MC38 + E. coli.MO: splenocytes + MC38 + oxaliplatin.MOE: splenocytes + MC38 + oxaliplatin + E .coli).(A) The populations of the three APCs (B cells, DCs, and macrophages) in the CD45+ cells.(B) The MHC-II + populations of the three APCs in the CD45 + cells.(C) The CD80 + /CD86 + double positive populations of the three APCs in the CD45+ cells.(D) The CD25+ population in CD8 + T cells.(E) Percentage of the dead MC38-EGFP cells after receiving each treatment in a spleenocyte co-culture system.Statistical differences between among groups were computed using one-way ANOVA with Turkey's multiple comparison test and differences were considered statistically significance when p-value is < 0.05 (n = 3 for each group).

Figure R6 .
Figure R6.Assesment of CCK-8 signals for murine spleenocytes.(A) Standard curve demostrating colleration between spleenocyte counts with the CCK-8 signals (A450).Spleenocytes were counted using hemocytometer under microscope prior to the generation of standard curve.(B) Raw absorbance data for the MC38-spleenocyte co-culture assay.In the co-culture assay, 5 x 10 4 spleenocytes were added to each
; 5a-e; 6a-b.o Please note that n=2 in figures 3b, c, e, f, i, j; 4c-d.o Please note that the figure legend style does not comply with the journal guidelines i.e. all the figure legends are in a run-on style.o Please note that a separate 'Data Information' section is required in the legends of all the figures.o Please define the annotated p values ****/***/**/* in the legend of figures 1c, g; 2c, f; 3a-b, d-j; 4a-c, f-h; 5a-e; 6a-b as appropriate.-Limit keywords to max 5. -Please add callout for Fig 1H, 2H and 3F.

I
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CD8 T-cells, and macrophages were among the immune cell types that bound to the surface of the MC38 cells.(FigureS16in the manuscript's supplementary data).
statistical methods and measures: -are tests one-sided or two-sided?-are there adjustments for multiple comparisons?-exact statistical test results, e.g., P values = x but not P values < x; -definition of 'center values' as median or average; -definition of error bars as s.d. or s.e.m.
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Hemolysis assay of mice RBCs incubated with E.coli (DH5)
Figure R1.Safety assessment of systemic E. coli administration in mice models.(A) Body weight changes (percentage relative to the same mice prior E. coli IV administration) of mice received E. coli treatment.A total of 4 doses (1 x 10 8

7 )
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